Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
  • A61K51/0487—Metallocenes, i.e. complexes based on a radioactive metal complexed by two cyclopentadienyl anions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. proteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/083—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins the peptide being octreotide or a somatostatin-receptor-binding peptide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
  • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F17/00—Metallocenes

Definitions

• the present invention concerns compounds and methods of labeling peptides or other molecules with 18 F or 19 F or any other suitable radionuclide o use, for example, in PET or NMPv in vivo imaging.
• a targeting molecule such as a protein or a peptide is linked to a substituted metal locene complex which is reacted with the 18 F or 19 F shortly before performing the PET or NMR in vivo imaging on the patient.
• the labeled molecule may be used for targeting a cell, tissue, organ or pathogen to be imaged or detected.
• Exemplary targeting molecules include, but are not limited to, an antibody, antigen- binding antibody fragment, bispecific antibody, affibody, diabody, minibody, ScFvs, aptamer, avimer, targeting peptide, somatostatin, bombesin, octreotide, RGD peptide, folate, folate analog or any other molecule known to bind to a disease-associated target.
• F-labeled molecules of high specific activity may be prepared in a very short time and are suitable for use in imaging techniques. Labeling may occur in a saline medium suitable for direct use in vivo.
• the labeled molecules are stable under physiological conditions, although for certain purposes, such as kit formulations, a stabilizing agent such as ascorbic acid, trehalose, sorbitol or mannitoi may be added.
• PET Positron Emission Tomography
• FDG fluoro-D-glucose
• WO 2011/095150 Al discloses conjugates of 18 F carriers having bioactive organic compounds and their methods of preparation.
• the carrier comprises a metaliocene complex fixed on a solid support, preferably via a phosphine linker.
• metaliocene complex is fixed onto the solid support, then F is added to replace the original metal protective groups, typically CI, w ith 18 F. Then the T ions and the excess of 18 F are 18
• a targeting molecule is added to the F-labelcd
• the agents comprise a protected Staudinger component that is convertible into unprotected Staudinger component before conjugation reaction, and is linked with carrier by spacer group.
• the present invention concerns compounds and methods relating to
• M is a metal selected from the group consisting of titanium, zirconium and hafnium;
• X 1 and X 2 which can be the same or different, are a halogen atom, a R ⁇ OR', OCOR', SR', NR' 2 or PR'2 group, wherein the R' substituents are linear or branched, saturated or unsaturated C1-C20 alkyi, C3-C20 cycloaikyi, C6-C 2 o a yl, C 7 -C 2 o aikylaryi, C 7 -C 20 arylaikyi radicals, optionally containing one or more heteroatoms belonging to the groups 1 3- 1 7 of the Periodic Table of the Elements;
• X 1 and X 2 can also be interconnected via a cyclic structure of ⁇ 40 atoms comprising one or more of C, N, O, F, Si, B, P.
• R 1 , R “ , R ⁇ R 4 , R ⁇ R 6 , R 7 , R 8 and R 9 are H or linear or branched, saturated or unsaturated C1-C20 alkyl, C3-C20 cycloaikyi, C 6 -C 2 o aryl, C 7 -C 2 o aikylaryi, C 7 -C 2 o arylaikyi radicals, optionally containing one or more heteroatoms belonging to the groups 1 3- 17 of the Periodic Table of the Elements.
• Two or more adjacent R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 groups on a cyclopentadienyl residue may be connected by a saturated, or unsaturated bridge containing up to 40 atoms comprising one or more o C, N, S, O, F, Si, B, P. These bridges may contain or be part of up to 5 carbo- or hetero-cycies.
• R 1 , R 2 , R ⁇ R 4 , R 5 , R", R 7 , R 8 and R 9 of different cyclopentadienyl residues may be connected by a saturated or unsaturated bridge containing up to 40 atoms comprising C, N, S, O, F, Si, B, P. These bridges may contain or be part of up to 5 carbo- or hetero-cycles.
• R is selected from the group consisting of F, CH 2 F, CHF 2 , CF 3 , OCF 3 ;
• linker moiety L may be protected at its functional end with a protecting group, as known in the art.
• a protective group is advantageous for some linkers to facilitate purification of intermediates or inhibit side reactions on undesired functionalities.
• a selection of protective group may be found in Theodora Greene et al. Protective groups in synthesis, John Wiley & Sons, Inc, New York, Chichester, Weinheim, Brisbane, Toronto, Singapore, 1999.
• the tert-butyl group may act as a protective group for a carboxylic acid group.
• Many protective groups are known, their choice depends on which group is to be protected and on the protection conditions.
• the metallocene complex of Formula (I) above is then attached v ia the linker group I. to a targeting molecule A to form a conjugate of
• A is a targeting molecule or recognition element known to bind to a disease-associated target.
• targeting molecule and “recognition element” are used interchangeably in the present description.
• delivery molecules primarily concern peptide moieties, many other types o delivery molecules, such as oligonucleotides, hormones, growth factors, cytokines, chemokines, angiogenic factors, anti-angiogenic factors, immunomodulators, proteins, nucleic acids, antibodies, antibody fragments, drugs, interleukins, interferons, oligosaccharides, polysaccharides, siderophores,
• lipids, etc. may be F-labeled and utilized for imaging purposes.
• the click chemistry involves the reaction of a targeting molecule such as an antibody or antigen-binding antibody fragment, comprising a functional group such as an alkyne, nitrone or an a/ide group, with a metallocene comprising the corresponding reactive moiety such as an azide, alkyne or nitrone.
• a targeting molecule such as an antibody or antigen-binding antibody fragment
• a metallocene comprising the corresponding reactive moiety such as an azide, alkyne or nitrone.
• the targeting molecule comprises an alkyne
• the chelating moiety or carrier will comprise an azide, a nitrone or similar reactive moiety.
• the click chemistry reaction may occur in vitro to form a highly stable, targeting molecule that, after having been labelled with a radionuclide, is then administered to a subject.
• conjugates of Formula (II) are labeled w ith a radionuclide Y via a fast exchange reaction of X 1 and X 2 .
• Y 1 and Y " is a radionuclide, the same or different.
• molecules that bind directly to receptors such as somatostatin, octreotide, bombesin, folate or a folate analog, an ROD peptide or other known receptor ligands may be labeled and used for imaging.
• Receptor targeting agents may include, for example, TA 138, a non-peptide antagonist for the integrin ⁇ ⁇ 3 receptor ( Liu et ai., 2003, Bioconj. Chem. 14: 1052-56).
• Other methods of receptor targeting imaging using metal chelates are known in the art and may be utilized in the practice of the claimed methods (see, e.g., Andre et ai., 2002, J. Inorg. Biochem. 88: 1 -6; Pearson et al., 1996, J. Med., Chem. 39: 1 361 -71 ).
• the type o diseases or conditions that may be imaged is limited only by the availability o a suitable delivery molecule for targeting a cell or tissue associated with the disease or condition.
• a suitable delivery molecule for targeting a cell or tissue associated with the disease or condition.
• Many such delivery molecules are known.
• any protein or peptide that is known in the art are known.
• proteins or peptides may include, but are not limited to, antibodies or antibody fragments that bind to tumor-associated antigens (TAAs). Any known TAA-binding antibody or fragment may be
• labeled with F by the described methods and used for imaging and/or detection of tumors, for example by PET scanning or other known techniques.
• labeling with the radionuclide is carried out in the liquid phase.
• the labeled conjugate is then purified and utilized for imaging purposes in the patient. Purification is carried out preferably v ia HPLC since most of the hospitals with PET imaging equipments have access to HPLC systems suitable to be used with radioactive compounds. Thus, the specialized radio-chemist in the hospital receives the
• Formula (II) and Formula (III) can facilitate the diagnosis of a disease.
• the complex of Formula (I) can be conjugated with a wide variety of recognition elements (A) selective for a certain diseases or subtype thereo .
• Suitable conjugation chemistry may be chosen among the various forms of "Click chemistry" which have been previously discussed, or the Staudinger reaction (Staudinger, FL; Meyer, J. Helv. Chim. Acta 1919, 2, 635 64) performed even on solid phase. (Scabini, M. WO 201 1 /095 1 50 A l : Kim H. Org. Lett (2006) 8, 1 149- 1 1 5 1 )
• FIG. 1 is a scheme showing the preparation and use of the compounds according to an embodiment of the invention. DETAILED DESCRIPTION
• a "peptide” refers to any sequence of naturally occurring or non-naturally occurring amino acids of between 2 and 100 amino acid residues in length, more preferably between 2 and 10, more preferably between 2 and 6 amino acids in length.
• An "amino acid” may be an L-amino acid, a D-amino acid, an amino acid analogue, an amino acid derivativ e or an amino acid mimetic.
• pathogen includes, but is not limited to fungi, viruses, parasites and bacteria, including but not limited to human immunodeficiency v irus ( H IV ), herpes virus, cytomegalov irus, rabies virus, influenza v irus, hepatitis B virus, Sendai v irus, feline leukemia virus.
• Reovirus polio v irus, human serum parv o-like virus, simian virus 40, respiratory syncytial v irus, mouse mammary tumor v irus, Varicella-Zoster virus.
• Streptococcus agalactiae Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilus influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis and Clostridium tetani.
• the metal M is preferably titanium or zirconium, more preferably titanium.
• R 1 , R 2 , R ⁇ R 4 , R 5 , R 6 , R 7 , R 8 and R 9 , hich can be the same or different, are preferably selected from the group consisting of H, metyl, benzyl, isobutyl, tert-butyi, methoxy, trimetylsilyl, 1 ,3 -butadiene- 1 ,4-diyl, as represented below:
• R 1 , R 2 , R ⁇ R 4 , R 5 , R 6 , R ⁇ R 8 and R 9 are more preferably H, methyl, or benzyl.
• X 1 and X 2 are preferably selected from F, CI, Br, I, alkynes, OR" or SR", wherein R" is a divalent radical selected from the group consisting of C6-C20 a ylidene being either unsubstituted or substituted with a group comprising 2-40 C, N, O, F, Si, B, P atoms.
• Preferred alkynes are:
• groups X 1 and X 2 are carboxylate, thiocarboxyiate, dithiocarboxyiate, carbamate, thiocarbamate, dithiocarbamate, trithiocarbamate, thiophosphinate dithiophosphinate, thiophosphonate, dithiophosphonate, trithiophosphonate, thiophosphate, dithiophosphate, trithiophosphate, tetrathiophosphate, sulfonate, thiosulfonate.
• groups X 1 and X 2 are heterocycies such as imidazole, triazoie, tetrazol, thiazol, benzimidazoi, benzotriazoi, indole, indazoie, pyrazole, pyrrol i don, azetan, azepine, benzodiazepine, pipeidine, purin, morpholine, piperazine, triazine, oxazoie, hydantoine, aziridine, pyrrolidine, pyrrole, hexamethyeneimine, azaindoies or alkylated, acylated, aryiated, annulated or heteroannulated members of this group having pKa ⁇ 1 2.
• heterocycies such as imidazole, triazoie, tetrazol, thiazol, benzimidazoi, benzotriazoi, indole, indazoie, pyrazole,
• X 1 and X 2 can also be interconnected via a cyclic structure of ⁇ 40 atoms comprising one or more of C, N, O, F, Si, B, P.
• X 1 and X 2 which can be the same or different, are more preferably benzene- 1 ,2-dithiolate, ethanethioiate, benzoate, 3,3-dimethyl-l-butyne, chlorine, fluorine, pentasuifide.
• L is a linker selected from the group consisting of:
• R is selected from the group consisting of F, CH 2 F, CHF 2 , CF 3 , OCF 3 ;
• Preferably 4 ⁇ m ⁇ 12, more preferably m 6.
• the linker group L comprises an arylene moiety incorporated in the linker structure, typically a phenyiene moiety, and/or a cyclooctyne terminal group.
• a metallocene complex of Formula (I) is used to prepare a conjugate with a targeting molecule or recognition element A v ia the linker group L.
• the substituent L or linker group L, bears a terminal functional group that can react with a corresponding functional group of a targeting molecule A, so that a compound or conjugate of Formula (II) can be formed:
• Preferred terminal groups for the linker L are phosphine, azide, alkene, alkyne, carboxylic acid, carboxylic ester, phosphoramidite, 11-phosphonatealdchyde, ketone, chloroimidate, thiol, or a chelator that is able to complex an ion together with the molecule A.
• a particularly preferred linker group L is -CH 2 -Ar-COO- bearing a terminal group as defined above.
• Another particularly preferred linker group L is
• R is selected from the group consisting of F, CH 2 F, CHF 2 , CF 3 , (XT;:
• Ar is C6-C20 aryl, C7-C20 aikyiaryl, or C7-C20 arylalkyl radical, optionally containing more heteroatoms belonging to the groups 13-17 of the Periodic Table of the Elements.
• the synthesis begins with a cyclopentadiene bearing from 0 to 5 alkyl substituents, R 5 -R 9 .
• This compound exists as a readily interconvertible mixture of tautomers.
• the mixture of tautomers is then deprotonated using a strong base such as BuLi, sec-BuLi or tert-Bu Li, but not necessarily a Li-base.
• the resulting Lithium cyclopenrtadienide is then transmetallated with TiCl. ⁇ to give a cyclopentadienyl trichloro titanium complex.
• L is suitably protected to avoid unwanted reactions with the cyclopentadienyl titanium trichloride or the Li-cyclopentadienide.
• the product of this reaction is a titanocenyl dichioride bearing two cyclopentadienyl residues, one with the groups R 1 -R 5 and the other with the groups R 6 -R 9 and L.
• the protective group of the linker L is then removed to allow conjugation with the recognition element.
• conjugates of Formula (II) may be prepared from metalloeenc compounds of Formula (I) and targeting molecules A using the click chemistry technology.
• the click chemistry approach was originally conceived as a method to rapidly generate comple substances by joining small subunits together in a modular fashion.
• Chemoselective ligation such as the Staudinger ligation (azide-phosphine) can also be used to prepare conjugates of Formula ( I I ) from metallocene compounds of Formula (I) and targeting molecules A.
• Staudinger ligation azide-phosphine
• the Staudinger chemistry has the best efficiency and compatibility for live-cell labeling and mass spectrometry (MS) applications for biological research.
• Staudinger reaction occurs between a methyl ester phosphine (P3) and an azide (N3) to produce an aza-ylide intermediate that is trapped to form a stable covending bond.
• P3 methyl ester phosphine
• N3 azide
• Staudinger ligation The chemical biology application is now known as Staudinger ligation. Unlike typical crosslinking methods used in biological research, this reaction chemistry depends upon a pair of unique reactive groups that are specific to one another and also foreign to biological systems. Because phosphines and azides do not occur in cells, they react only with each other in biological samples, resulting in minimal background and few artifacts. This is the meaning of "chemoseiective”.
• Reactive targeting molecule may be formed by either chemical conjugation or by biological incorporation.
• the targeting molecule such as an antibody or antibody fragment, may be activated with an azido moiety, a substituted cyclooctyne or alkyne group, or a nitrone moiety.
• the targeting molecule comprises an azido or nitrone group
• the corresponding targetable construct will comprise a substituted cyclooctyne or alkyne group, and vice versa.
• Such activated molecules may be made by metabolic incorporation in liv ing cells, as discussed above.
• the moiety labeled with F or other diagnostic and/or therapeutic agents may comprise a peptide or other targetable construct.
• Labeled peptides or proteins
• RGD peptide, octreotide, bombesin or somatostatin may be selected to bind directly to a targeted cell, tissue, pathogenic organism or other target for imaging, detection and/or diagnosis.
• labeled peptides may be selected to bind indirectly, for example using a bispecilic antibody with one or more binding sites for a targetable construct peptide and one or more binding sites for a target antigen associated with a disease or condition.
• Bispeciflc antibodies may be used, for example, in a pretargeting technique wherein the antibody may be administered first to a subject. Sufficient time may be allowed for the bispeci fic antibody to bind to a target antigen and for unbound antibody to clear from circulation. Then a targetable construct, such as a labeled peptide, may be administered to the subject and allowed to bind to the bispecific antibody and localize at the diseased cell or
• F-labeled targetable constructs may be determined by PET scanning or other known techniques.
• targetable constructs can be of diverse structure and are selected not only for the availability o an antibody or fragment that binds with high affinity to the targetable construct, but also for rapid in vivo clearance when used within the pre-targeting method and bispecific antibodies (bsAb) or multispecific antibodies.
• Hydrophobic agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance.
• hydrophilic chelating agents to offset the inherent hydrophobicity of many organic moieties.
• sub-units of the targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobic and some of which are hydrophilic. Aside from peptides, carbohydrates may also be used.
• Peptides having as few as two amino acid residues, preferably two to ten residues, may be used and coupled to metallocene complexes substituted with suitable linker groups.
• the linker should be a low molecular weight moiety, preferably having a molecular weight of less than 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000 daltons or 5,000 daltons. More usually, the targetable construct peptide will have four or more residues.
• the targetable construct may also comprise unnatural amino acids, e.g., D-amino acids, in the backbone structure to increase the stability of the peptide in vivo.
• unnatural amino acids e.g., D-amino acids
• other backbone structures such as those constructed from non-natural amino acids or peptoids may be used.
• the peptides used as targetable constructs are conveniently synthesized on an automated peptide synthesizer using a solid-phase support and standard techniques of repetitive orthogonal deprotection and coupling. Free amino groups in the peptide, that are to be used later for conjugation of chelating moieties or other agents, are advantageously blocked with standard protecting groups such as a Boc group, while N-terminal residues may be acetylated to increase serum stability. Such protecting groups are well known to the skilled artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). Antibodies
• Targeting antibodies of use may be specific to or selective for a variety of cell surface or disease-associated antigens.
• Exemplary target antigens of use for imaging or treating various diseases or conditions such as a malignant disease, a cardiovascular disease, an infectious disease, an inflammatory disease, an autoimmune disease, a metabolic disease, or a neurological (e.g..
• ne urodege nera t i v e ) disease may include a-fetoprotein (AFP), A3, amyloid beta, CA 125, colon-specific antigen-p (CSAp), carbonic anhydrase IX, CCCL19, CCCL21 , CSAp, GDI, CD la, CD2, CD3, CD4, CD5, CDS, CD11A, CD 14, CD 15, CD 16, CD 18, CD 19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD 126, CD 133, CD 138, CD 147, CD 154, CXCR4, CXCR7, CXCL12, II IF- la.
• AFP a-fetoprotein
• HCG human chorionic gonadotropin
• HER-2/neu HER-2/neu
• H GB-1 hypoxia inducible factor
• IP- 10 IP- 10, KS-1, Le(y), low-density lipoprotein (I.DL), MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5a-c, MUC16, NCA- 95, NCA-90, as NF- ⁇ , pancreatic cancer mucin, PAM4 antigen, placental growth factor, p53, PLAGL2, Prl, prostatic acid phosphatase, PSA, FRAME, PSMA, PIGF, tenascin, RANTES, T 101, TAC. TAG72, TF.
• MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include a finity chromatography with Protein-A or Protein-G Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, for example, Coligan at pages 2.7.1 -2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et al.. "Purification of Immunoglobulin G (IgG)," in METHODS I MOLECULAR BIOLOGY, VOL. 10, pages 79- 104 (The Humana Press, Inc. 1992). After the initial raising of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art, as discussed below. Chimeric Antibodies
• a chimeric antibody is a recombinant protein in which the variable regions of a human antibody hav e been replaced by the variable regions of for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody.
• Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject.
• CDRs complementarity-determining regions
• Hybridoma 13:469 ( 1 994), produced an I .L2 chimera by combining DNA sequences encoding the VK and VH domains o murine 1.1.2, an anti-CD22 monoclonal antibody, with respective human ⁇ and IgGl constant region domains.
• a chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heav y and light v ariable chains o the mouse immunoglobulin into the corresponding v ariable domains o a human antibody.
• the mouse framework regions ( FR ) in the chimeric monoclonal antibody are also replaced with human FR sequences. As simply transferring mouse CDRs into human FRs often results in a reduction or ev en loss o antibody a finity, additional modification might be required in order to restore the original a finity o the murine antibody.
• the phage display technique may be used to generate human antibodies
• Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005).
• the advantage to constructing human antibodies from a diseased indiv idual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
• transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols.
• Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al., Nature Genet. 7: 13 ( 1 994), Lonberg et al., Nature 368:856 (1994), and Taylor et al, Int. Immun. 6:579 ( 1994).
• a non-limiting example of such a system is the XenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231 : 1 1- 23, incorporated herein by reference) from Abgenix ( Fremont, Calif ).
• the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
• the targeting molecules of use for imaging, detection and/or diagnosis may incorporate any antibody or fragment know n in the art that has binding specificity for a target antigen associated with a disease state or condition.
• Such known antibodies include, but are not limited to, liR 1 (anti-IGF- 1 R, U.S. patent application Ser. No. 12/772,645, filed Mar. 12, 201 0) hPAM4 (anti-pancreatic cancer mucin, U.S. Pat. No. 7,282,567), hA20 (anti-CD2(), U.S. Pat. No. 7.25 1 , 164 ), hA 19 (anti-CD 1 9, U.S. Pat. No.
• hIMMU31 anti-AFP, U.S. Pat. No. 7,300,655
• hLLl anti-CD74, U .S. Pat. No. 7,3 1 2,3 1 8
• hi.1.2 anti-CD22, U.S. Pat. No. 7.074.403
• hMu-9 anti-CSAp, U.S. Pat. No. 7.387,773
• hl .243 anti-HLA-DR. U.S. Pat. No. 7.612, 1 80).
• hMN-14 anti-CEACAM5, U.S. Pat. No. 6,676.924)
• hMN- 1 5 anti-CEACAM6, U.S. Pat. No.
• Type Culture Collection (ATCC, Manassas, Va. ).
• ATCC Manassas, Va.
• the skilled person will realize that antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search o the ATCC, NCBI and or US PTC) databases for antibodies against a selected disease-associated target of interest.
• the antigen binding domains of the cloned antibodies may be amplified, excised, iigated into an expression vector, transfected into an adapted host ceil and used for protein production, using standard techniques well known in the art.
• Antibody fragments which recognize specific epitopes can be generated by known techniques.
• the antibody fragments are antigen binding portions o an antibody, such as F(ab')2, Fab', F(ab)2, Fab, Fv. sFv and the like.
• F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule and Fab' fragments can be generated by reducing disulfide bridges o the F(ab')2 fragments.
• Fab' expression libraries can be constructed (1 1 use et al., 1 989, Science. 246: 1 274- 128 1 ) to allow rapid and easy identi ication o monoclonal Fab' fragments with the desired specificity.
• An antibody fragment can be prepared by proteolytic hydrolysis of the lull length antibody or by expression in E. coli or another host o the DNA coding for the fragment.
• Goidenberg U.S. Pat. Nos. 4,036,945 and 4,331 ,647 and references contained therein, which patents are incorporated herein in their entireties by re erence.
• any o the antibodies, antibody fragments or antibody fusion proteins described herein may be conjugated to a metallocene complex of Formula (I) to form an immunoconjugate o Formula ( I I ).
• Methods for covalent conjugation of substituted metallocene complexes having linker moieties L are described abov e.
• Affibodies are small proteins that function as antibody mimetics and are o use in binding target molecules.
• a fibodies were developed by combinatorial engineering on an alpha helical protein scaffold (Nord et al., 1995. Protein Eng 8:601 -8: Nord et al., 1 997. Nat Biotechnol 1 5:772-77).
• the affibody design is based on a three helix bundle structure comprising the IgG binding domain of protein A (Nord et al., 1995; 1997).
• Affibodies with a wide range o binding affinities may be produced by randomization of thirteen amino acids involved in the Fc binding activity of the bacterial protein A (Nord et al., 1 995; 1997). After randomization, the PCR amplified library was cloned into a phagemid vector for screening by phage display of the mutant proteins.
• affibodies may be used as targeting molecules in the practice of the claimed methods and compositions. Labeling with metal-conjugated 18F having linker moieties L may be performed as described above. Affibodies are commercially available from A ffibody AB (Solna, Sweden).
• binding peptides may be produced by phage display methods that are well known in the art.
• peptides that bind to any of a variety of disease-associated antigens may be identified by phage display panning against an appropriate target antigen, cell, tissue or pathogen and selecting for phage with high binding affinity.
• Various methods of phage display and techniques for producing diverse populations of peptides are ell known in the art. For example, U.S. Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, disclose methods for preparing a phage library.
• the phage display technique involves genetically manipulating bacteriophage so that small peptides can be expressed on their surface (Smith and Scott, 1 985, Science 228: 13 1 5- 13 1 7; Smith and Scott, 1993, Meth. Enzymol. 2 1 :228-257).
• a targeting molecule may comprise an aptamer.
• Methods of constructing and determining the binding characteristics of aptamers are well known in the art. For example, such techniques are described in U.S. Pat. Nos. 5.582,98 1 , 5,595,877 and 5.637,459.
• Aptamers may be prepared by any known method, including synthetic, recombinant, and purification methods, and may be used alone or in combination with other ligands specific for the same target. In general, a minimum of approximately 3 nucleotides, preferably at least 5 nucleotides, are necessary to effect specific binding. Aptamers of sequences shorter than 10 bases may be feasible, although aptamers of 10, 20, 30 or 40 nucleotides may be preferred.
• Aptamers need to contain the sequence that confers binding specificity, but may be extended with flanking regions and otherwise derivati/ed.
• the binding sequences of aptamers may be flanked by primer-binding sequences, facilitating the amplification of the aptamers by PGR or other amplification techniques.
• the flanking sequence may comprise a specific sequence that preferentially recognizes or binds a moiety to enhance the immobilization of the aptamer to a substrate.
• Aptamers may be isolated, sequenced, and/or amplified or synthesized as conventional DNA or R A molecules.
• aptamers of interest may comprise modi ied oligomers. Any of the hydroxy! groups ordinarily present in aptamers may be replaced by phosphonate groups, phosphate groups, protected by a standard protecting group, or activated to prepare additional linkages to other nucleotides, or may be conjugated to solid supports.
• the technique generally involves selection from a mixture of candidate aptamers and step-wise iterations of binding, separation of bound from unbound aptamers and amplification. Because only a small number of sequences (possibly only one molecule of aptamer) corresponding to the highest affinity aptamers exist in the mixture, it is generally desirable to set the partitioning criteria so that a significant amount of aptamers in the mixture (approximately 5- 50%) is retained during separation. Each cycle results in an enrichment of aptamers with high affinity for the target. Repetition for between three to six selection and amplification cycles may be used to generate aptamers that bind with high affinity and specificity to the target. Avimers
• the targeting molecules may comprise one or more avimer sequences.
• Avimers are a class of binding proteins somewhat similar to antibodies in their affinities and specificities for various target molecules. They were developed from human extracellular receptor domains by in vitro ex on shuffling and phage display (Silverman et al., 2005, Nat. Biotechnol. 23: 1493-94; Silverman et al., 2006. Nat. Biotechnol. 24:220).
• the resulting multidomain proteins may comprise multiple independent binding domains, that may exhibit improved affinity (in some cases sub-nanomolar) and specificity compared with single-epitope binding proteins.
• Additional details concerning methods of construction and use of avimers are disclosed, for example, in U.S. Patent Application Publication Nos. 20040175756, 20050048512, 20050053973, 20050089932 and 20050221384.
• the conjugate of Formula (II) can also be prepared by reacting the recognition element A in the solid state with the complex of Formula (I).
• An example is a peptide like octreotide, which is synthesized on solid phase and may be attached to the complex of Formula (I), while still being bound to the synthesis resin. Subsequently, it may be released from the solid phase as conjugate of Formula (II).
• Other applications may include proteins or oligonucleotides either synthesized on a synthesis support or isolated bound to some reactive affinity matrix.
• Conjugates of Formula ( I I ) can be labeled with F via a fast F exchange to prepare a labeled conjugate of Formula ( I I I ):
• Y 1 and Y 2 is a radionuclide which, can be introduced to displace X 1 and X 2 by direct bond with M, as it is preferably done with 18 F (1 10m), 120 I (81.0 m), 124 I (4. 1 8 d), or 76 Br (16.2 h). The displacement is performed immediately prior to usage to minimize loss of labeling due to radioactive decay.
• Y is preferably 18 F.
• labeling with the radionuclide is carried out in the liquid phase.
• the introduction of the F tracer with a hal li e of 1 10 min. has to proceed rapidly, i.e. in less than three half-lives.
• the labeling temperature is below 1 00°C.
• the preferred labeling temperature is low enough to avoid irreversible damage of the tracer conjugate.
• the positive counterion ( ' -ion) is generally complexed with suitable complexing agents, such as the cryptands commercially av ailable under the tradename "Kryptofix" (Merck KGAA) to obtain dissolution.
• the high radioactivity involved in the introduction of the tracer requires special safety measures to protect the laboratory personnel.
• the labeling process has to meet these requirements by comprising as few manipulations as possible.
• many solvent exchanges reactions at or above boiling point of the solvent or reactions under pressure should be avoided or minimized since they pose a great risk of contamination. Therefore one pot procedures, possibly using immobilized reagents, are preferred (Langstrom, B. et al, Acta Chem, Scand. 1999, 53, 651 ; Miller, P. M. et al. Angew. Chem. Int. Ed. 2008, 47, 8998).
• the waste, often containing excess radiolabel must be in the most concentrated form to allow safe disposal, separate from non-radioactive wastes. (C. S. Elmore. Ann. Rep. Med. Chem. 2009, 44, 515).
• the processes may even be performed on a conjugate of Formula (II) immobilized on a solid phase.
• Preferred stationary phases are C-18 analogous phases based on polystyrene, polyacrylamide or polypropylene and can be obtained from commercial sources even in SPE-cartridge form.
• a specialized radio-chemist in the hospital receives the metallocene-targeting molecule conjugate of Formula (II) and loads it onto cartridge containing a stationary phase as described above. 18
• the progress of the thiol-lluoride exchange may be v isually monitored by the color change (in case of a displacement o benzene dithiol groups by fluoride, the titanocene absorbance maximum of 450 and 600nm disappears. This is valid for all compounds with a cyclopentadienyl titanium chromophore.
• the F-iabeled molecules may be formulated to obtain compositions that include one or more pharmaceutically suitable excipients, one or more additional ingredients, or some combination of these. These can be accomplished by known methods to prepare pharmaceutically useful
• activ e ingredients i.e., the F-labeled molecules
• one or more pharmaceutically suitable excipients Sterile phosphate-bu fered saline is one example of a pharmaceutically suitable excipient.
• Other suitable excipients are well known to those in the art. See, e.g., Ansel et al, PHARMACEUTICAL DOSAGE FOR S AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1 990), and Gennaro (ed.), REM INGTON'S PHARMACEUTICAL SCIENCES, 1 8th Edition ( Mack Publishing Company 1990), and revised editions thereof.
• compositions described herein are parenteral injection.
• Injection may be intravenous, intraarterial, intralymphatic, intrathecal, subcutaneous or intracav itary (i.e., parenterally).
• parenteral administration the compositions will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with a pharmaceutically acceptable excipient.
• excipients are inherently nontoxic and nontherapeutic. Examples of such excipients are saline. Ringer's solution, dextrose solution and Hank's solution.
• Nonaqueous excipients such as fixed oils and ethyl oleate may also be used.
• a preferred excipient is 5% dextrose in saline.
• the excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.
• Other methods of administration, including oral administration, are also contemplated.
• compositions comprising F-iabeled molecules can be used for intravenous administration via, for example, bolus injection or continuous infusion.
• Compositions for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
• Compositions can also take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and or dispersing agents.
• the compositions can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen- ree water, before use.
• compositions may be administered in solution.
• the pH of the solution should be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5.
• the formulation thereo should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate or citrate and the like.
• the bu fer is potassium biphthalate ( KHP), which may act
• the formulated solution may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM.
• a stabilizing agent such as glycerol, albumin, a globulin, a detergent, a gelatin, a protamine or a salt of protamine may also be included.
• the compositions may be administered to a mammal subcutaneousiy, intravenously, intramuscularly or by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses.
• the dosage of F label to administer will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history.
• a saturating close of the 1 8F-labeled molecules is administered to a patient.
• the dosage may be measured by millicuries. A typical range for 18F imaging studies would be live to 10 mCi.
• F-labeled peptides to be administered to a subject may occur by any route known in the art, including but not limited to oral, nasal, buccal, inhalational, rectal, vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial,
• F-labeled peptides are administered in a pretargeting protocol
• the peptides would preferably be administered i.v. Imaging Using Labeled Molecules
• kits containing components suitable for imaging, diagnosing and/or detecting diseased tissue in a patient using labeled compounds.
• exemplary kits may contain an antibody, fragment or fusion protein, such as a bispecific antibody of use in pretargeting methods as described herein.
• Other components may include a targetable construct for use with such bispecific antibodies.
• the targetable construct is pre-conjugated to a metallocene
• a targetable construct may be attached to one or more di ferent radionuclide, as described above.
• a device capable of delivering the kit components may be included.
• the kit components may be packaged together or separated into two or more containers.
• the containers may be v ials that contain sterile, lyophili/ed formulations of a composition that are suitable for reconstitution.
• a kit may also contain one or more buffers suitable for reconstitution and/or dilution o other reagents.
• Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterile ly within the containers. Another component that can be included is instructions to a person using a kit for its use.
• reaction mixture was stirred for 18 hours at room temperature. Progress of the reaction was monitored via thin layer chromatography (TLC) (hexane-AcOEt 8:2 ). Upon completion the reaction was terminated by adding NallCO; (lg).
• the raw reaction product was purified on a silica gel column (25 g) using hexane-diethyl ether
• Step 2 Preparation of t-butyl 4-(cyclopentadiene-l-vlmethyl) benzoate
• the obtained raw product was purified on a column of silica gel (30g) using a mixture of hexane - diethyl ether (9: 1) as eluent.
• Step 3 Preparation of ( ⁇ 4 - [(tert butoxy) carbonyl] phenyl ⁇ methyl) ( ⁇ 5-2 ,4- cyclopentadiene-l-yl) 7 dichlorocyclopentadienyltitanium
• the ester t-butyl 4-(cyclopentadiene- 1 -ylmethyl ) benzoate prepared according to Step 2 above (1.18 mmoles, 300 mg) was dissolved in anhydrous THF (2ml ). The solution was put under static atmosphere of argon and brought to -78°C. A solution of di tert-butyl litium (1.5 M) in hcxane (0.820 ml, 1 ,23 mmoles, 1 .04 cqv) was added very slowly and the resulting solution was kept at -78°C for 2h.
• ⁇ - M R (200 MHz) : ⁇ 7.98 (d,2H), 7.25 (d,2H), 6.51-6.30 (m,9H), 4.20 (s,2H), 2.98-2.90 (d,2H), 1.59 (s,9H).
• Step 4 Preparation of ( ⁇ 4 - [(tert butoxy) carbonyl] phenyl ⁇ methyl) ( ⁇ 5-2 ,4- cyclopentadiene-l-yl)] 1 ,2-benzendithiolate cyclopentadienyltitanium
• Step 3 Synthesis of ( ⁇ [4-(tert-butoxycarbonyl)phenyl]methyl ⁇ cyclopentadienyl) dichloro(pent methylcyclopentadienyl) titanium
• Step 1 and 2 were performed as in Example 1 .
• Step 3 was performed in the following way:
• Step 4 ( ⁇ [ 4-(tert-butoxycarbonyl)phenyl]methyl ⁇ cyclopentadienyl) benzenedithiolate (pentamethylcvclopentadienyl) titanium
• reaction was performed as described in Example 1 , step 4, starting from ( ⁇ [4-(tert- butoxycarbonyl)phenyl]methyl ⁇ cyclopentadienyl) dichloro(pentamethylcyclopentadienyl) titanium; i.e. reaction of the alkylated titanocene dichloride with benzenedithiol.
• Example 3a De-protection of t-butvl ester to obtain the corresponding acid.
• Example 3b De-protection of t-butvl ester to obtain the corresponding acid.
• reaction mixture was allowed to come to room temperature and was allowed to stir for 3 hours. Tic-analysis of the reaction mixture (TLC DCM-AcOEt 9:1, v/v) analysis indicated complete conversion.
• the product was characterized further by NMR: ( !
• HOBt 1-hydroxy-benzotriazole
• EDCI N-(3-dimethylaminopropyl)-N-ethylcarbodiimide chlorhydrate
• Example 6 above was carried out with 19 F.
• ⁇ - MR (200MHz) : ⁇ 6.10 (d,d, 111) ; ⁇ 3.80 (m, 111); ⁇ 3.45 (t, 211); 62.64 (m, 111); ⁇ 2.10 (m, 211); ⁇ 1.60 (m.1411)
• the reaction was monitored by TLC (hexane 100% as eluent), quenched by addition of a saturated solution of a ⁇ SO, (3ml) and the phases were separated. The aqueous phase was extracted with Et 2 0 and combined organic layers were dried over Na ⁇ SO.; , filtered and concentrated under vacuum. The obtained raw product was purified using a column of silica gel (40g) and He.xane as eluent to obtain

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